Female eutherian mammals have evolved a mechanism by which the dosage of functional Xlinked genes in each somatic cell is equalized to that of males. This dosage compensation is accomplished by transcriptionally inactivating genes on one of the two X chromosomes in females. Thus, for most X-linked genes in female somatic cells, an active and inactive allele reside within the same nucleus but are differentially regulated and expressed. The long-term goal of this project is to determine the mechanism of X chromosome inactivation. This proposal will specifically investigate the molecular basis for maintaining the differential expression of genes on the active versus the inactive X chromosome in somatic cells by examining the role of DNA-protein interactions within specific X-linked genes. In vivo footprinting of intact cells will be used to identify sequence-specific DNA-binding proteins which interact with either the active or inactive allele of the human and mouse HPRT genes. Such DNA-binding proteins are likely to have a direct role in the differential expression of these genes on the active and inactive X chromosomes. The 5' region of each gene will be studied in hybrid cell lines which permit separate analysis of the active, inactive, and reactivated alleles. Similar studies will also be carried out on normal human and mouse cells. In addition, cytosine methylation in the 5' region from both genes will be determined in vivo by genomic sequencing. Together, these studies will examine in vivo the correlation between binding of sequence-specific proteins, DNA methylation, and transcriptional activity of genes on the active and inactive X chromosomes. DNAprotein interactions specific to either the active or inactive allele of the HPRT genes will be further characterized in vitro by gel mobility-shift assays, DNase I footprinting, and partial purification of the binding protein(s). These in vitro studies will include an examination of the interaction of the binding proteins with other mammalian promoters (both autosomal and X-linked), and an analysis of the evolutionary conservation of the binding activity(s) in heterologous mammalian extracts. Studies of DNA-protein interactions correlated with differential expression of the active and inactive alleles of specific X-linked genes should provide significant insight into the chromosome-wide mechanism for coordinate gene regulation by X chromosome inactivation.